Advances in physiological activities and synthesis of β-nicotinamide mononucleotide / 生物工程学报

Nicotinamide mononucleotide (NMN) is one of the key precursors of coenzyme Ⅰ (NAD+). NMN exists widely in a variety of organisms, and β isomer is its active form. Studies have shown that β-NMN plays a key role in a variety of physiological and metabolic processes. As a potential active substance in anti-aging and improving degenerative and metabolic diseases, the application value of β-NMN has been deeply explored, and it is imminent to achieve large-scale production. Biosynthesis has become the preferred method to synthesize β-NMN because of its high stereoselectivity, mild reaction conditions, and fewer by-products. This paper reviews the physiological activity, chemical synthesis as well as biosynthesis of β-NMN, highlighting the metabolic pathways involved in biosynthesis. This review aims to explore the potential of improving the production strategy of β-NMN by using synthetic biology and provide a theoretical basis for the research of metabolic pathways as well as efficient production of β-NMN.

Advances on microbial synthesis of L-proline and trans-4-hydroxy-L-proline / 生物工程学报

L-proline (L-Pro) is the only imino acid among the 20 amino acids that constitute biological proteins, and its main hydroxylated product is trans-4-hydroxy-L-proline (T-4-Hyp). Both of them have unique biological activities and play important roles in biomedicine, food and beauty industry. With the in-depth exploration of the functions of L-Pro and T-4-Hyp, the demand for them is gradually increasing. Traditional methods of biological extraction and chemical synthesis are unable to meet the demand of "green, environmental protection and high efficiency". In recent years, synthetic biology has developed rapidly. Through the intensive analysis of the synthetic pathways of L-Pro and T-4-Hyp, microbial cell factories were constructed for large-scale production, which opened a new chapter for the green and efficient production of L-Pro and T-4-Hyp. This paper reviews the application and production methods of L-Pro and T-4-Hyp, the metabolic pathways for microbial synthesis of L-Pro and T-4-Hyp, and the engineering strategies and advances on microbial production of L-Pro and T-4-Hyp, aiming to provide a theoretical basis for the "green bio-manufacturing" of L-Pro and T-4-Hyp and promote their industrial production.

New targeted compounds-biosynthesis of phytocannabinoids / 生物工程学报

Phytocannabinoids are bioactive terpenoids that are exclusive to Cannabis sativa L. The main pharmacologically active phytocannabinoids are Δ9-tetrahydrocannabinol and cannabidiol, both target endogenous cannabinoid receptors. Δ9-tetrahydrocannabinol and cannabidiol have extensive therapeutic potential due to their participation in many physiological and pathological processes in human body by activating the endocannabinoid system. At present, Δ9-tetrahydrocannabinol, cannabidiol and their analogues or combination preparations are used to treat epilepsy, vomiting in patients with cancer chemotherapy, spasticity in multiple sclerosis and relieve neuropathic pain and pain in patients with advanced cancer. With the further exploration of the application value of Δ9-tetrahydrocannabinol and cannabidiol as well as the increasing demand for standardization of pharmaceutical preparations, it is imminent to achieve large-scale production of Δ9-tetrahydrocannabinol and cannabidiol in the pharmaceutical industry. In this article, pharmacological research progress of phytocannabinoids in recent years, biosynthetic pathways of phytocannabinoids and the mechanism of key enzymes as well as various product development strategies of cannabinoids in pharmaceutical industry are reviewed. By exploring the potential of synthetic biology as an alternative strategy for the source of phytocannabinoids, it will provide a theoretical basis for the research and development of microbial engineering for cannabinoids synthesis, and promote the large-scale production of medicinal cannabinoids.

Exploring the role and mechanisms of diallyl trisulfide and diallyl disulfide in chronic constriction-induced neuropathic pain in rats

Background@#Garlic oil is a rich source of organosulfur compounds including diallyl disulfide and diallyl trisulfide. There have been studies showing the neuroprotective actions of these organosulfur compounds. However, the potential of these organosulfur compounds in neuropathic pain has not been explored. The present study was aimed at investigating the pain attenuating potential of diallyl disulfide and diallyl trisulfide in chronic constriction injury (CCI)-induced neuropathic pain in rats. The study also explored their pain-attenuating mechanisms through modulation of H2S, brain-derived neurotrophin factor (BDNF) and nuclear factor erythroid 2-related factor 2 (Nrf2). @*Methods@#The rats were subjected to CCI injury by ligating the sciatic nerve in four places. The development of neuropathic pain was measured by assessing mechanical hyperalgesia (Randall–Selittotest), mechanical allodynia (Von Frey test), and cold allodynia (acetone drop test) on 14th day after surgery. @*Results@#Administration of diallyl disulfide (25 and 50 mg/kg) and diallyl trisulfide (20 and 40 mg/kg) for 14 days led to a significant reduction in pain in CCI-subjected rats. Moreover, treatment with these organosulfur compounds led to the restoration of H2S, BDNF and Nrf2 levels in the sciatic nerve and dorsal root ganglia. Coadministration of ANA-12 (BDNF blocker) abolished pain attenuating actions as well as BDNF and the Nrf2 restorative actions of diallyl disulfide and diallyl trisulfide, without modulating H2S levels. @*Conclusions@#Diallyl disulfide and diallyl trisulfide have the potential to attenuate neuropathic pain in CCI-subjected rats possibly through activation of H2S-BDNF-Nrf2 signaling pathway.

Reinforcement of Rhodobacter sphaeroides cofactor NADPH to increase the production of farnesol / 生物工程学报

Farnesol (FOH) is produced by dephosphorylation of farnesyl diphosphate (FPP) derived from two universal building blocks, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In Rhodobacter sphaeroides these building blocks are generated by MEP pathway, however, many of the biosynthetic reactions and biotransformations in the MEP pathway are limited by low availability of NADPH. Improvement of the amount of intracellular NADPH may enhance the synthesis of FOH. In this study, we utilized the strategies of increasing the production of NADPH and decreasing the consumption of NADPH. The expression of glucose 6-phosphate isomerase (pgi) and glutamate dehydrogenase (gdhA) were inhibited by RNA interference, respectively, and overexpression of 6-glucose phosphate dehydrogenase (zwf) and 6-glucose phosphate dehydrogenase (gnd) in the pentose phosphate pathway were carried out. The results showed that the content of NADPH in the recombinant strains increased significantly, the highest FOH production of RSpgii in the RNA interfered strain was 3.91 mg/g, and the FOH production increased to 3.43 mg/g after zwf gene and gnd gene has been overexpressed. In order to obtain strains with higher FOH production, we used RSpgii as the starting strain, and zwf, gnd and co-overexpressed zwf + gnd gene were overexpressed in RSpgii, respectively. The highest FOH production of the strain RSzgpi reached to 4.48 mg/g which was 2.24 times that of the starting strain RS-GY2.

High-level production of glucose oxidase by recombinant Pichia pastoris using a combined strategy / 生物工程学报

To enhance the production of glucose oxidase by recombinant Pichia pastoris, two strategies were developed, which were namely co-feeding of methanol and sorbitol and co-expressing of the protein disulfide isomerase (PDI) and Vitreoscialla hemoglobin (VHb). The volumetric activity reached 456 U/mL by using the strain X33/pPIC9k-GOD, in 5 liter fermentator, with the co-feeding of methanol and sorbitol, it was 0.2 fold higher than that only feeding by methanol. The improved strain was obtained by co-expressing PDI-VHb with GOD. While fermented in a 5 liter fermentator by feeding methanol and sorbitol, the activity of the improved strain reached 716 U/mL with a yield of 7 400 mg/L total soluble protein concentration. These results indicated that heterologous protein expression level can be enhanced by optimizing fermentation condition and co-expression molecular chaperon in Pichia pastoris.

Accuracy of Corvis ST for the measurement of central corneal thickness and intraocular pressure in myopia / 中华实验眼科杂志

Background Corvis ST corneal biomechanical analyzer (Corvis ST) can offer corneal biomechanical parameters,intraocular pressure (IOP) and central corneal thickness (CCT),and measured IOP value was corrected based on CCT and biomechanical factors.Corvis ST is applied abroad,but the study on its accuracy is few in China.Objective This diagnostic trial was to evaluate the accuracy of Corvis ST for CCT and IOP measurement in myopic population.Methods Fifty-six eyes from 56 myopic patients were prospective recruited in Visual Science and Optometry Center of Guangxi from November to December in 2012.IOP was measured by using Corvis ST and Goldmann applanation tonometer (GAT),and CCT was measured by Corvis ST and A type ultrasonic pachymetry.The CCT difference between Corvis ST and A type ultrasonic pachymetry as well as IOP between Corvis ST and GAT were compared by using paired-t test,and agreements of measured outcomes were analyzed by Bland-Altman method.This study was approved by the Ethic Committee of People's Hospital of Guangxi and written informed consent was obtained from all subjects.Results The CCT from Corvis ST was (539.82± 19.79) μm,which was significantly higher than (535.34± 19.41) μm from A type ultrasonic pachymetry (t =4.19,P＜0.001).Bland-Altman analysis revealed that the CCT measured by Corvis ST was 4.5 μm higher than that of A type ultrasonic pachymetry,with the 95％ limit of agreement ranged from-11.2 to 20.2 μm,and 7.1％ (4/56) of points were located at the outside of the 95％ confidence interval.The IOP measured by Corvis ST and GAT were (15.75±1.60) mmHg and (16.23 ±2.40) mmHg,respectively,showing statistically significant difference between the two methods (t=2.15,P =0.04).Bland-Altman analysis revealed that the IOP measurement of Corvis ST was 0.5 mmHg lower than that of GAT,with the 95％ limit of agreement ranged from-3.8 to 2.8 mmHg,and 3.57％ (2/56) of points were located at the outside of the 95％ confidence interval.Conclusions CCT obtained by Corvis ST is higher than that by A type ultrasonic pachymetry with poor agreement between these two outcomes,and the two methods cannot replace each other clinically in myopic eyes.IOP value from Corvis ST is slightly lower than that from GAT,showing a good agreement between these two outcomes.The IOP value of Corvis ST shows satisfactory accuracy.

Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1 / 生物工程学报

The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.

Construction of a recombinant Escherichia coli for high trehalose production / 生物工程学报

Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.

Impact of sulfentanyl on ropivacaine epidural block during abdominal panhysterectomy / 实用医学杂志

Objective To explore the impact of sulfentanyl on sufentanil epidural block during abdominal panhysterectomy. Methods 90 patients scheduled for panhysterectomy were randomly divided into three groups. Tthe control group received epidural administration of 1% ropivacaine of 0.2 mL/kg after 2% idocaine of 3 mL , while the study group 1 received 10μg sufentanil and the study group 2 received 20μg sufentanil in addition to the medications used in the control group. The anesthetic effect, changes in vital signs, and incidence of adverse reactions were compared among the three groups. Results In group S1 and group S2, the onset of epidural anesthesia was faster , time to the highest plane of sensory blockade and time to degree 3 in the Bromag scores were faster , duration of sensory blockade was longer , and OAA/S score was better , as compared with group D , with significant statistical significances (P<0.01);and the effect was better in group S2 than in group S1. There was no difference among the three groups in adverse reactions. MAP , HR and SPO2 were lower in groups S1 and S2 than in group D during the procedure, with a statistical difference (P<0.05). Conclusions Proper dose of sufentanil plays a positive role in ropivacaine epidural block during panhysterectomy , not only increases the onset of anesthesia, but also makes the anesthestic effect better, and has higher safety It is worth popularizing clinically.

Construction of Saccharomyces cerevisiae mutant with knockout of SNF4 gene / 生物工程学报

Construction and ethanol production effects of SNF4 gene knockout in Saccharomyces cerevisiae were described in this paper. For knockout of SNF4 gene in S. cerevisiae YS2, a PCR-amplified disruption cassette was used, encoding the short flanking homologous regions to the SNF4 gene and Kan(r) as selectable marker. The SNF4 gene disruption cassette was transformed into S. cerevisiae YS2 through LiAc/SS Carrier DNA/PEG. The positive transformants were grown on G418 plates and verified by PCR. The Kan(r) marker was rescued by transforming plasmid pSH65 into positive transformants and inducing expression of Cre recombinase in galactose-containing medium. Lastly, the YS2-deltaSNF4 strain, in which SNF4 allele gene were completely knocked out, was obtained by repeating the same procedure. The result of anaerobic fermentation showed that ethanol production of the SNF4 gene knockout strain had increased by 7.57 percent as compared with the original strain YS2. The experiment indicated ethanol production could be improved significantly with the approach ofSNF4 gene knockout by Cre-LoxP system.

Construction of Aspergillus niger lipase mutants with oil-water interface independence / 生物工程学报

Based on previous bioinformational analysis results, two Aspergillus niger lipase (ANL) mutants, ANL-Ser84Gly and ANL-Asp99Pro were constructed to screen ANL mutants with oil-water interface independence. ANL-Ser84Gly still displayed a pronounced interfacial activation, while ANL-Asp99Pro displayed no interfacial activation. The specific activity of ANL-Ser84Gly towards p-nitrophenyl palmitate (-myristate, -laurate and -decanoate) decreased by 29.8% (53.1, 60.1 and 77.1, respectively) than that of ANL, while the specific activity of ANL-Asp99Pro towards p-nitrophenyl palmitate increased by 2.2-fold. The mutation in the hinge region at both sides of the lid domain also destabilized various secondary structure factors of ANL-S84G and ANL-D99P, which resulted in a substantial decrease in thermostability. The achievement to construct oil-water interface-independent ANL mutants would help to further understand lipase interfacial activation mechanism.

Expression of a pectin lyase A gene from Aspergillus niger in Pichia pastoris GS115 / 生物工程学报

In this study, the mature peptide sequence of a pectin lyase gene A was amplified from Aspergillus niger strain EIM-6 by using RT-PCR reverse transcription technique. The cloned gene was then inserted into a Pichia pastoris expression vector pPIC9k to produce the recombinant expression plasmid pPIC9K-pelA. By using electric shocks, we successfully transformed the recombinant pPIC9K-pelA into Pichia pastoris GS115. The activity of the engineered strain reached to 2.3 U/mL after induction with the final concentration of 1.5% methanol. SDS-PAGE analysis revealed that the pPIC9K-pelA transformant had an additional protein band of approximately 38 kD, which was not present in the control. There were no significant differences between the recombinant and native pectin lyase with regard to their hydrolysis activities.

A Rapid and Efficient Method for Directed Screening of Lipase-producing Burkholderia cepacia Complex with Organic Solvent Tolerance from Plant Rhizosphere / 微生物学通报

Lipase from Burkholderia cepacia complex is one of the most versatile biocatalyst and is used widely in many biotechnological application fields including detergent additives, the resolution of racemic compounds, etc. Based on the known whole genomic information of B. cepacia, both ampicillin and kanamycin were added to the TB-T media, the traditional selective media, to screen B. cepacia complex strains from rhizosphere soil samples. The single colonies on the plates with the modified TB-T media were then qualitatively determined the ability to produced the extracellular lipase in the rhodamine B-olive oil agar plates. Thirty-five strains of lipolytic pseudo-B. cepacia complex were isolated and the positive rate of lipolytic bacteria was 65%. Among them, 15 pseudo-B. cepacia complex strains with the tolerance to benzene, n-hexane and n-heptane at the concentration of 10% (V/V) were selected and identified by the recA gene sequence. All of the 15 lipolytic bacteria belonged to the B. cepacia complex.

Hemostatic Effect of Reptilase on Radical Gastrectomy for Gastric Cancer / 中国药房

OBJECTIVE:To observe the haemostasis outcome of treating radical gastrectomy for gastric cancer with Rep-tilase.METHODS:A total of30patients underwent radical gastrectomy for gastric cancer were randomly divided into reptilase group and control group,the preoperative and postoperative blood clotting functional parameter changes of the2groups were observed and the volumes of intraoperative blood loss and blood infusion were recorded.RESULTS:There was no significant difference in the blood clotting functional parameters before and after operation in the reptilase group,the intraoperative blood loss volume and blood infusion volume in this group were all lower than in the control group(P

STUDY ON THE NUTRITIONAL VALUE OF MORTIERELLA ISABELINA AND ITS EFFECT ON REGULATION OF BLOOD LIPIDS IN RATS / 营养学报

Objective: 1.To analyze the nutritional composition and evaluation of the nutritional value of Mortierella isabelina powder. 2.To investigate the effects of Mortierella isabelina powder on prevention and regulation of hyperlipidemia in rats. Methods: 1. Mycelium powder was analyzed for proteins, lipids polysaccharides, fibre, ash, vitamin E, amino acids, fatty acids and minerals. 2. Mycelium powder was used to feed rats in different dosages together with high lipid diet for 10 days. 3. Mycelium powder was used to feed hyperlipidemic rats in different dosages for 30 days. Results: (1) There were about 20% of proteins and more than 50% of lipids in the mycelium. Further examination found that the mycelium contained rich essential amino acids and unsaturated fatty acids. The mycelium also contained vitamin E and useful minerals such as K, Ca, P, Fe, Zn and Mn. (2) 0.6 or 1.2 g/(kg?d) dosage of mycelium powder could prevent the rise of serum lipids. (3) When the mycelium powder was used to feed hyperlipidemic rats, the serum cholesterol, triglyceride, LDL C and VLDL C of the rats could be reduced, meanwhile HDL C could be increased. Conclusion: The results indicated that Mortierella isabelina powder has potential nutritional value and can be used to regulate the blood lipids in hyperlipidemic rats.